System, method, and product for dynamic noise reduction in scanning of biological material

ABSTRACT

Systems and methods are described for processing an emission signal, such as a fluorescent signal, to compensate for noise in an excitation beam, such as a laser beam. As one example, a scanning system is described that includes an excitation signal generator that provides an excitation signal having one or more representative excitation values representative of an excitation beam; an excitation reference provider that provides at least one excitation reference value; a normalization factor generator that compares the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and a comparison processor that adjusts at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/304,092 filed Nov. 25, 2002, now U.S. Pat. No. 6,813,567 which is acontinuation of U.S. application Ser. No. 09/683,216 filed 09/683,216filed Dec. 3, 2001, now U.S. Pat. No. 6,490,533 issued Dec. 3, 2002which claims priority from U.S. Provisional Patent Application Ser. No.60/286,578, filed Apr. 26, 2001. The entire disclosure and contents ofthe above patents and applications are hereby incorporated by reference.The present application is related to a U.S. patent application entitled“System, Method, and Product for Pixel Clocking in Scanning ofBiological Materials,” and to a U.S. patent application entitled“System, Method, and Product for Symmetrical Filtering in Scanning ofBiological Materials,” both of which are filed concurrently herewith andboth of which are hereby incorporated by reference herein in theirentireties for all purposes.

BACKGROUND

1. Field of the Invention

The present invention relates to scanning systems for examiningbiological material and, in particular, to noise reduction in opticalscanning systems having a laser to excite fluorescently taggedbiological materials.

2. Related Art

Synthesized nucleic acid probe arrays, such as Affymetrix® GeneChip®synthesized probe arrays, have been used to generate unprecedentedamounts of information about biological systems. For example, acommercially available GeneChip® array set from Affymetrix, Inc. ofSanta Clara, Calif., is capable of monitoring the expression levels ofapproximately 6,500 murine genes and expressed sequence tags (EST's).Experimenters can quickly design follow-on experiments with respect togenes, EST's, or other biological materials of interest by, for example,producing in their own laboratories microscope slides containing densearrays of probes using the Affymetrix® 417™ or 427™ Arrayers or otherspotting devices. Analysis of data from experiments with synthesizedand/or spotted probe arrays may lead to the development of new drugs andnew diagnostic tools.

In some conventional applications, this analysis begins with the captureof fluorescent signals indicating hybridization of labeled targetsamples with probes on synthesized or spotted probe arrays. The devicesused to capture these signals often are referred to as scanners. Due tothe relatively small emission signals sometimes available from thehybridized target-probe pairs, the presence of background fluorescentsignals, the high density of the arrays, variations in theresponsiveness of various fluorescent labels, and other factors, caremust be taken in designing scanners to properly acquire and process thefluorescent signals indicating hybridization. For example, U.S. Pat. No.6,171,793 to Phillips, et al., hereby incorporated herein in itsentirety for all purposes, describes a method for scanning probe arraysto provide data having a dynamic range that exceeds that of the scanner.Nonetheless, there is a continuing need to improve scanner design toprovide more accurate and reliable fluorescent signals and thus provideexperimenters with more sensitive and accurate data.

SUMMARY OF THE INVENTION

Systems, methods, and products to address these and other needs aredescribed herein with respect to illustrative, non-limiting,implementations. Various alternatives, modifications and equivalents arepossible. For example, certain systems, methods, and computer softwareproducts are described herein using exemplary implementations foranalyzing data from arrays of biological materials produced by theAffymetrix® 417™ or 427™ Arrayer. Other illustrative implementations arereferred to in relation to data from Affymetrix® GeneChip® probe arrays.However, these systems, methods, and products may be applied withrespect to many other types of probe arrays and, more generally, withrespect to numerous parallel biological assays produced in accordancewith other conventional technologies and/or produced in accordance withtechniques that may be developed in the future. For example, thesystems, methods, and products described herein may be applied toparallel assays of nucleic acids, PCR products generated from cDNAclones, proteins, antibodies, or many other biological materials. Thesematerials may be disposed on slides (as typically used for spottedarrays), on substrates employed for GeneChip® arrays, or on beads,optical fibers, or other substrates or media. Moreover, the probes neednot be immobilized in or on a substrate, and, if immobilized, need notbe disposed in regular patterns or arrays. For convenience, the term“probe array” will generally be used broadly hereafter to refer to allof these types of arrays and parallel biological assays.

In accordance with one preferred embodiment, a method is described thatincludes the steps of (1) directing an excitation beam to a plurality ofpixel locations on a substrate; (2) determining one or morerepresentative excitation values, each related to a value of theexcitation beam as directed to at least one of the plurality of pixellocations; (3) detecting an emission signal having one or more emissionvalues; (4) correlating each of the one or more emission values with oneor more of the representative excitation values; (5) providing at leastone excitation reference value; (6) comparing the excitation referencevalue to at least one representative excitation value, therebygenerating a normalization factor; and (7) adjusting at least oneemission value based, at least in part, on the normalization factor. Oneor more probes of a biological microarray may be disposed in relation tothe substrate; for example, probes may be coupled to the substrate. Theone or more probes may be disposed at different probe locations on asurface of the substrate. In some implementations, the substrate mayinclude different polymer sequences coupled to the surface of thesubstrate. The different polymer sequences may include differentoligonucleotide sequences, wherein each of the different polymersequences is coupled in a different probe location of the surface. Insome implementations, each of the probe locations has an area ofone-hundredth of a square centimeter or less. Also, in someimplementations, the substrate may include more than one thousanddifferent ligands of known sequence collectively occupying an area ofless than one square centimeter, the different ligands occupyingdifferent known locations within the area.

In accordance with further implementations of these preferredembodiments, step (2) includes directing the excitation beam to adichroic mirror, and determining the representative excitation valuesbased on a partial excitation beam that passes through the dichroicmirror. Also, the emission signal may arise from the direction of theexcitation beam to the plurality of pixel locations. As used in thiscontext, the word “arise” is intended to have a broad meaning so as toencompass various cause and effect relationships wherein the directingof the excitation beam causes or results in, directly or indirectly, theemission signal. As just one non-limiting example, the excitation beammay be a laser beam directed to a location on the substrate wherefluorescently labeled receptors are disposed, and the emission signalmay be a fluorescent signal resulting from excitation of those labeledreceptors. In these and other implementations, step (4) may includespatially correlating the emission values with the representativeexcitation values. Also in these and other implementations, step (2) mayinclude determining a first representative excitation value related to apower of the excitation beam as directed to a first of the plurality ofpixel locations; step (3) may include detecting an emission valuearising from the direction of the excitation beam to the first pixellocation; and step (4) may include correlating the first emission valuewith the first representative excitation value. In accordance withvarious embodiments, the excitation reference value may be based, atleast in part, on at least one of the one or more representativeexcitation values, and/or on a plurality of representative excitationvalues related to values of the excitation beam as directed to pixellocations in one or more scan lines.

The method, in accordance with some embodiments, may further include thestep of (8) filtering the representative excitation values to provideone or more filtered representative excitation values. In theseembodiments, the excitation reference value is based, at least in part,on at least one of the one or more filtered representative excitationvalues. In these and other embodiments, the excitation reference valuemay be based, at least in part, on a measured calibration value and/oron a predetermined specification value.

In accordance with yet other embodiments, a system is described forprocessing an emission signal having one or more emission values. Thesystem includes an excitation signal generator that provides anexcitation signal having one or more representative excitation valuesrepresentative of an excitation beam. The system also has an excitationreference provider that provides at least one excitation referencevalue; a normalization factor generator that compares the excitationreference value to at least one representative excitation value, therebygenerating a normalization factor; and a comparison processor thatadjusts at least one emission value corresponding to the at least onerepresentative excitation value based, at least in part, on thenormalization factor. The excitation reference value may be determinedbased at least in part on a low-frequency component of the excitationsignal. The at least one representative excitation value may include, asexamples, an instantaneous analog value or a sampled digital value. Insome implementations, the comparison processor adjusts the at least oneemission value corresponding to the at least one representativeexcitation value based on multiplying or dividing the emission value bythe normalization factor. The excitation signal may include a lasersignal, and the emission signal may include a fluorescent signalresulting from excitation of a fluorophore by the laser signal.

In accordance with a further embodiment, a method is described forprocessing an emission signal having one or more emission values. Themethod includes providing at least one excitation reference value;comparing the excitation reference value to at least one excitationvalue, thereby generating a normalization factor; and adjusting at leastone emission value corresponding to the at least one excitation valuebased, at least in part, on the normalization factor.

A scanning system is described in accordance with some embodiments. Thesystem includes one or more excitation sources that generate one or moreexcitation beams; an excitation signal generator that provides anexcitation signal having one or more representative excitation valuesrepresentative of the excitation beam; an excitation reference providerthat provides at least one excitation reference value; a normalizationfactor generator that compares the excitation reference value to atleast one representative excitation value, thereby generating anormalization factor; and a comparison processor that adjusts at leastone emission value corresponding to the at least one representativeexcitation value based, at least in part, on the normalization factor.The scanning system may include a processor and a memory unit, whereinthe normalization factor generator includes a set of normalizationfactor generating instructions stored in the memory unit and executed incooperation with the processor. The comparison processor may alsoinclude a set of comparison processing instructions stored in the memoryunit and executed in cooperation with the processor.

A computer program product is described with respect to otherembodiments. The product includes a set of normalization factorgenerating instructions stored in a memory unit of a computer andexecuted in cooperation with a processor of the computer. Theseinstructions are constructed and arranged to compare at least oneexcitation reference value to at least one representative excitationvalue, thereby generating a normalization factor. The product alsoincludes a set of comparison processing instructions stored in thememory unit and executed in cooperation with the processor, constructedand arranged to adjust at least one emission value corresponding to theat least one representative excitation value based, at least in part, onthe normalization factor. The at least one emission value results fromexcitation of a labeled receptor at a probe location of a probe array.

A method for analyzing molecules is described with respect to someembodiments. The method includes (1) directing an excitation beam to aplurality of pixel locations on a surface having a plurality of probelocations, each probe location including one or more probe molecules;(2) determining one or more representative excitation values, eachrelated to a value of the excitation beam as directed to at least one ofthe plurality of pixel locations; (3) detecting an emission signalhaving one or more emission values; (4) correlating each of the one ormore emission values with one or more of the representative excitationvalues; (5) providing at least one excitation reference value; (6)comparing the excitation reference value to at least one representativeexcitation value, thereby generating a normalization factor; (7)adjusting at least one emission value based, at least in part, on thenormalization factor; and (8) analyzing at least one probe locationbased, at least in part, on the at least one adjusted emission value.

The above embodiments and implementations are not necessarily inclusiveor exclusive of each other and may be combined in any manner that isnon-conflicting and otherwise possible, whether they be presented inassociation with a same, or a different, embodiment or implementation.The description of one embodiment or implementation is not intended tobe limiting with respect to other embodiments and/or implementations.Also, any one or more function, step, operation, or technique describedelsewhere in this specification may, in alternative implementations, becombined with any one or more function, step, operation, or techniquedescribed in the summary. Thus, the above embodiment and implementationsare illustrative rather than limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings. In the drawings, like reference numerals indicatelike structures or method steps and the leftmost digit of a referencenumeral indicates the number of the figure in which the referencedelement first appears (for example, the element 160 appears first inFIG. 1). In functional block diagrams, rectangles generally indicatefunctional elements and parallelograms generally indicate data. Inmethod flow charts, rectangles generally indicate method steps anddiamond shapes generally indicate decision elements. All of theseconventions, however, are intended to be typical or illustrative, ratherthan limiting.

FIG. 1 is a simplified graphical representation of an arrangement ofscanner optics and detectors suitable for providing excitation andemission signals for processing by a noise compensation module such asshown in FIG. 4 or 9;

FIG. 2A is a perspective view of a simplified exemplary configuration ofa scanning arm portion of the scanner optics and detectors of FIG. 1;

FIG. 2B is a top planar view of the scanning arm of FIG. 2A as it scansbiological features on one embodiment of a probe array being moved by atranslation stage under the arm's arcuate path;

FIG. 3 is a functional block diagram of one embodiment of ascanner-computer system including a scanner having a noise compensationmodule such as shown in FIG. 4 or 9;

FIG. 4 is a functional block diagram of one embodiment of a noisecompensation module for providing an emission signal normalized tocompensate for noise in the excitation source;

FIG. 5A is a graphical representation of one embodiment of a probefeature showing bi-directional scanning lines such as may be implementedusing the scanning arm of FIGS. 2A and 2B;

FIG. 5B is an illustrative plot of pixel clock pulses aligned with thescanned probe feature of FIG. 5A to show illustrative radial positionsampling points;

FIG. 5C is an illustrative plot of sampled emission voltages alignedwith the pixel clock pulses of FIG. 5B;

FIG. 5D is an illustrative plot of sampled excitation voltages alignedwith the pixel clock pulses of FIG. 5B;

FIG. 6A is a graphical representation of a simulated input waveformincluding noise;

FIGS. 6B and 6C are graphical representations of output waveforms fromalternative embodiments of asymmetrical filters responsive to the inputwaveform of FIG. 6A;

FIGS. 6D and 6E are graphical representations of output waveforms fromalternative embodiments of symmetrical filters responsive to the inputwaveform of FIG. 6A;

FIG. 7A is a graphical representation of an input waveform plotted as afunction of pixel clock pulses;

FIG. 7B is a graphical representation of an output waveform from oneembodiment of an asymmetrical filter responsive to the input waveform ofFIG. 7A as a function of pixel clock pulses in a sequence determined byscanning in a first direction;

FIG. 7C is a graphical representation of an output waveform from theasymmetrical filter of FIG. 7B responsive to the input waveform of FIG.7A as a function of pixel clock pulses in a sequence determined byscanning in a second direction opposite to the first direction;

FIG. 7D is a consolidated graphical representation of pixels shown inFIGS. 7B and 7C resulting from successive bi-directional scans of aprobe feature under asymmetrical filtering;

FIG. 7E is a graphical representation of the pixels of FIG. 7D shiftedso as to graphically compensate for the effects of phase delay;

FIG. 8A is a graphical representation of an input waveform plotted as afunction of pixel clock pulses;

FIG. 8B is a graphical representation of an output waveform from oneembodiment of a symmetrical filter responsive to the input waveform ofFIG. 8A as a function of pixel clock pulses in a sequence determined byscanning in a first direction;

FIG. 8C is a graphical representation of an output waveform from thesymmetrical filter of FIG. 8B responsive to the input waveform of FIG.8A as a function of pixel clock pulses in a sequence determined byscanning in a second direction opposite to the first direction;

FIG. 8D is a consolidated graphical representation of pixels shown inFIGS. 8B and 8C resulting from successive bidirectional scans of a probefeature under symmetrical filtering;

FIG. 8E is a graphical representation of the pixels of FIG. 8D shiftedso as to graphically compensate for the effects of phase delay;

FIG. 9 is a functional block diagram of one embodiment of a noisecompensation module for providing, together with a digital signalprocessing board and software on an associated computer, emission datanormalized to compensate for noise in the excitation source; and

FIG. 10 is a functional block diagram of one embodiment of aspects of acomputer of the scanner-computer system of FIG. 3 suitable forgenerating normalized emission signal data.

DETAILED DESCRIPTION

The description below is designed to present preferred embodiments andnot to be construed as limiting in any way. Also, reference will be madeto articles and patents to show general features that are incorporatedinto the present disclosure. Many scanner designs may be used in orderto provide excitation and emission signals appropriate for processing bynoise compensation module 310, which is described in detail below. Inreference to the illustrative implementation of FIG. 1, the term“excitation beam” refers to light beams generated by lasers. However,excitation sources other than lasers may be used in alternativeimplementations. Thus, the term “excitation beam” is used broadlyherein. The term “emission beam” also is used broadly herein. A varietyof conventional scanners detect fluorescent or other emissions fromlabeled target molecules or other material associated with biologicalprobes. Other conventional scanners detect transmitted, reflected, orscattered radiation from such targets. These processes are sometimesgenerally and collectively referred to hereafter for convenience simplyas involving the detection of “emission beams.” Various detectionschemes are employed depending on the type of emissions and otherfactors. A typical scheme employs optical and other elements to providean excitation beam, such as from a laser, and to selectively collect theemission beams. Also generally included are various light-detectorsystems employing photodiodes, charge-coupled devices, photomultipliertubes, or similar devices to register the collected emission beams. Forexample, a scanning system for use with a fluorescently labeled targetis described in U.S. Pat. Nos. 5,143,854 and 6,225,625, herebyincorporated by reference in their entireties for all purposes. Otherscanners or scanning systems are described in U.S. Pat. Nos. 5,578,832;5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096;6,185,030; 6,201,639; and 6,218,803 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Scanner Optics and Detectors 100.

FIG. 1 is a simplified graphical representation of illustrative scanneroptics and detectors (hereafter, simply “scanner optics”) 100. Scanneroptics 100 includes excitation sources 120A and 120B (generally andcollectively referred to as excitation sources 120). Any number of oneor more excitation sources 120 may be used in alternative embodiments.In the present example, sources 120 are lasers; in particular, source120A is a diode laser producing red laser light having a wavelength of635 nanometers and, source 120B is a doubled YAG laser producing greenlaser light having a wavelength of 532 nanometers. Further referencesherein to sources 120 generally will assume for illustrative purposesthat they are lasers, but, as noted, other types of sources, e.g., x-raysources, may be used in other implementations.

In the illustrated implementation, it is assumed that only one ofexcitation sources 120A and 120B is operational (in the sense ofgenerating an excitation beam 135) at any particular time. For example,source 120A and not source 120B may be operational for one arc scan byscanner optics 100, as described below, and source 120B and not source120A may be operational for a subsequent scan. Sources 120A and 120B mayalternate between successive scans, groups of successive scans, orbetween full scans of an array. For clarity, excitation beams 135A and135B are shown as distinct from each other in FIG. 1. However, inpractice, turning mirror 124 and/or other optical elements (not shown)typically are adjusted to provide that these beams follow the same path.Moreover, it also will be understood that the assumption that only onelaser is operational at a time is made only for the sake of convenienceand clarity of illustration. Implementations are contemplated thatinclude simultaneous operation of any number of excitation sources 120.Beams 135 in simultaneous operation typically, but need not, follow thesame path. Handbook of Biological Confocal Microscopy (James B. Pawley,ed.) (2.ed.; 1995; Plenum Press, NY), which includes information knownto those of ordinary skill in the art regarding the use of lasers andassociated optics, is hereby incorporated herein by reference in itsentirety.

Scanner optics 100 also includes excitation filters 125A and 125B thatoptically filter beams from excitation sources 120A and 120B,respectively. Filters 125 optionally are used to remove light atwavelengths other than the desired wavelengths, and need not be includedif, for example, sources 120A and 120B do not produce light at theseextraneous wavelengths. As noted, however, it may be desirable in someapplications to use inexpensive lasers and often it is cheaper to filterout-of-mode laser emissions than to design the laser to avoid producingsuch extraneous emissions.

The filtered excitation beams from sources 120A and 120B are combined inaccordance with any of a variety of known techniques. For example, oneor more mirrors, such as turning mirror 124, may be used to directfiltered beam from source 120A through beam combiner 130. The filteredbeam from source 120B is directed at an angle incident upon beamcombiner 130 such that the beams combine in accordance with opticalproperties techniques well known to those of ordinary skill in therelevant art. Most of combined excitation beams 135A and 135B (generallyand collectively referred to as beams 135) are reflected by dichroicmirror 136 and thence directed to periscope mirror 138 of theillustrative example. However, dichroic mirror 136 has characteristicsselected so that portions of beams 135A and 135B, referred torespectively as partial excitation beams 137A and 137B and generally andcollectively as beams 137, pass through it so that they may be detectedby excitation detector 110.

Detector 110 may be any of a variety of conventional devices fordetecting partial excitation beams 137, such as a silicon detector forproviding an electrical signal representative of detected light, aphotodiode, a charge-coupled device, a photomultiplier tube, or anyother detection device for providing a signal indicative of detectedlight that is now available or that may be developed in the future.Detector 110 generates excitation signal 194 that represents detectedpartial excitation beams 137A or 137B. In accordance with knowntechniques, the amplitude, phase, or other characteristic of excitationsignal 194 is designed to vary in a known or determinable fashiondepending on the power of excitation beam 135. The term “power” in thiscontext refers to the capability of beam 135 to evoke emissions. Forexample, the power of beam 135 typically may be measured in milliwattsof laser energy with respect to the illustrated example in which thelaser energy evokes a fluorescent signal. Thus, excitation signal 194has values that represent the power of beam 135 during particular timesor time periods.

In the illustrated example, excitation beams 135 are directed viaperiscope mirror 138 and arm end turning mirror 142 to an objective lens145. As described in greater detail below in relation to FIGS. 2A and2B, lens 145 in the illustrated implementation is a small, light-weightlens located on the end of an arm that is driven by a galvanometeraround an axis perpendicular to the plane represented by galvo rotation149 shown in FIG. 1. Objective lens 145 thus moves in arcs over asubstrate upon which biological materials have been synthesized or havebeen deposited. Flourophores associated with these biological materialsemit emission beam 152 (beam 152A in response to excitation beam 135A,and beam 152B in response to excitation beam 135B) at characteristicwavelengths in accordance with well known principles. Emission beam 152in the illustrated example follows the reverse path as described withrespect to excitation beam 135 until reaching dichroic mirror 136. Inaccordance with well known techniques and principles, thecharacteristics of mirror 136 are selected so that beam 152 (or aportion of it) passes through the mirror rather than being reflected.

In the illustrated implementation, filter wheel 160 is provided tofilter out spectral components of emission beam 152 that are outside ofthe emission band of the fluorophore. The emission band is determined bythe characteristic emission frequencies of those fluorophores that areresponsive to the frequency of excitation beam 135. Thus, for example,excitation beam 135A from source 120A, which is illustratively assumedto have a wavelength of 635 nanometers, excites certain fluorophores toa much greater degree than others. The characteristic emissionwavelength of a first illustrative fluorophore (not shown in FIG. 1)when excited by beam 135A is assumed to be 665 nanometers. Emission beam152A in this example typically will also include wavelengths above andbelow 665 nanometers in accordance with distributions that are known tothose of ordinary skill in the relevant art. Similarly, thecharacteristic emission wavelength of a second illustrative fluorophore(not shown in FIG. 1), when excited by beam 135A having a wavelength of532 nanometers, is illustratively assumed to be 551 nanometers. Thus,when excitation source 120A is operational, filter wheel 160 is turnedso that filter 162A is selected (typically under computer control) andwavelengths other than 665 nanometers are removed from filtered emissionbeam 154. Similarly, filter 162B is selected when source 120B isoperational so that wavelengths other than 551 nanometers are filteredout of beam 152B to produce filtered emission beam 154. In accordancewith techniques well known to those of ordinary skill in the relevantarts, including that of confocal microscopy, beam 154 may be focused byvarious optical elements such as lens 165 and also passed throughillustrative pinhole 167 or other element to limit the depth of field,and thence impinges upon emission detector 115.

Similar to excitation detector 110, emission detector 115 may be asilicon detector for providing an electrical signal representative ofdetected light, or it may be a photodiode, a charge-coupled device, aphotomultiplier tube, or any other detection device that is nowavailable or that may be developed in the future for providing a signalindicative of detected light. Detector 115 generates emission signal 192that represents filtered emission beam 154 in the manner noted abovewith respect to the generation of excitation signal 194 by detector 110.Emission signal 192 and excitation signal 194 are provided to noisecompensation module 310 for processing, as described below in relationto FIGS. 3 and 4.

FIG. 2A is a perspective view of a simplified representation of anillustrative scanning arm portion of scanner optics 100 in accordancewith this particular, non-limiting, implementation. Arm 200 moves inarcs around axis 210, which is perpendicular to the plane of galvorotation 149. A position transducer 215 is associated with galvanometer215 that, in the illustrated implementation, moves arm 200 inbi-directional arcs. Transducer 215, in accordance with any of a varietyof known techniques, provides an electrical signal indicative of theradial position of arm 200. Certain non-limiting implementations ofposition transducers for galvanometer-driven scanners are described inU.S. Pat. No. 6,218,803 to Montagu, et al., which is hereby incorporatedby reference in its entirety for all purposes. As described below, thesignal from transducer 215 is provided in the illustrated implementationto computer 350 so that clock pulses may be provided for digitalsampling of emission signals when arm 200 is in certain positions alongits scanning arc.

Arm 200 is shown in alternative positions 200′ and 200″ as it moves backand forth in scanning arcs about axis 210. Excitation beams 135 passthrough objective lens 145 on the end of arm 200 and excite fluorophoresthat may be contained in hybridized probe-target pairs in features 230on a substrate of probe array 240, as further described below. Thearcuate path of excitation beams 135 over probe array 240 isschematically shown for illustrative purposes as path 250. Emissionbeams 152 pass up through objective lens 145 as noted above. Probe array240 of this example is disposed on translation stage 242 that is movedin direction 244 so that arcuate path 250 repeatedly crosses the planeof probe array 240. As is evident, the resulting coverage of excitationbeams 135 over the plane of probe array 240 is therefore determined bythe footprint of beam, the speed of movement in direction 244, and thespeed of the scan. FIG. 2B is a top planar view of arm 200 withobjective lens 145 scanning features 230 on probe array 240 astranslation stage 242 is moved under path 250. As shown in FIG. 2B,arcuate path 250 of this example is such that arm 200 has a radialdisplacement of 0 in each direction from an axis parallel to direction244. For convenience of reference below, a direction 243 perpendicularto direction 244 is also shown in FIG. 2B. For illustrative purposes,direction 243 may hereafter be referred to as the “x” direction, anddirection 244 as the “y” direction.

Further details of confocal, galvanometer-driven, arcuate, laserscanning instruments suitable for detecting fluorescent emissions areprovided in PCT Application PCT/US99/06097 (published as WO99/47964) andin U.S. Pat. Nos. 6,185,030; 6,201,639; and 6,225,625, all of which havebeen incorporated by reference above.

Probe Array 240.

Probe array 240 as shown in FIGS. 2A and 2B is illustrative only and itwill be understood that numerous variations are possible with respect toproviding biological materials for scanning. For example, Affymetrix®GeneChip® arrays commercially available from Affymetrix, Inc., of SantaClara, Calif., referred to above, are synthesized in accordance withtechniques sometimes referred to as VLSIPS™ (Very Large ScaleImmobilized Polymer Synthesis) technologies. Probe arrays developed withthese technologies, and others that are now available and may in thefuture be developed for synthesizing arrays of biological materials, mayhereafter be referred to for convenience as “synthesized probe arrays.”This term refers generally to arrays in which probes have been built insitu on an array substrate, as contrasted, for example, to arrays inwhich pre-synthesized or pre-selected probes are deposited or positionedon or within a substrate.

Some aspects of VLSIPS™ technologies are described in the following U.S.Pat. No. 5,143,854 to Pirrung, et al.; U.S. Pat. No. 5,445,934 to Fodor,et al.; U.S. Pat. No. 5,744,305 to Fodor, et al.; U.S. Pat. No.5,831,070 to Pease, et al.; U.S. Pat. No. 5,837,832 to Chee, et al.;U.S. Pat. No. 6,022,963 to McGall, et al.; and U.S. Pat. No. 6,083,697to Beecher, et al. Each of these patents is hereby incorporated byreference in its entirety. The probes of these arrays typically consistof oligonucleotides that typically are synthesized by methods thatinclude the steps of activating regions of a substrate and thencontacting the substrate with a selected monomer solution. The regionsare activated with a light source shown through a mask in a mannersimilar to photolithographic techniques used in the fabrication ofintegrated circuits. Other regions of the substrate remain inactivebecause the mask blocks them from illumination. By repeatedly activatingdifferent sets of regions and contacting different monomer solutionswith the substrate, a diverse array of polymers is produced on thesubstrate. A variety of other techniques also exist for synthesizingprobe arrays. For example, U.S. Pat. Nos. 5,885,837 and 6,040,193describe the use of micro-channels or micro-grooves on a substrate, oron a block placed on a substrate, to synthesize arrays of biologicalmaterials.

As noted, techniques also exist for depositing or positioningpre-synthesized or pre-selected probes on or within a substrate orsupport. For convenience, probe arrays made in accordance with theseother techniques, or depositing/positioning techniques that may bedeveloped in the future, may hereafter be referred to as “spottedarrays.” Typically, spotted arrays are commercially fabricated onmicroscope slides. These arrays typically consist of liquid spotscontaining biological material of potentially varying compositions andconcentrations. For instance, a spot in the array may include a fewstrands of short polymers, such as oligonucleotides in a water solution,or it may include a high concentration of long strands of polymers, suchas complex proteins. The Affymetrix® 417™ and 427™ Arrayers are devicesthat deposit densely packed probe arrays of biological material on amicroscope slide in accordance with these techniques. Aspects of these,and other, spot arrayers are described in U.S. Pat. Nos. 6,121,048 and6,136,269, in PCT Applications Nos. PCT/US99/00730 (InternationalPublication Number WO99/36760) and PCT/US 01/04285, in U.S. patentapplication Ser. Nos. 09/122,216, 09/501,099, and 09/862,177, and inU.S. Provisional Patent Application Ser. No. 60/288,403, all of whichare hereby incorporated by reference in their entireties for allpurposes. Other techniques for generating spotted arrays also exist. Forexample, U.S. Pat. No. 6,040,193 to Winkler, et al. is directed toprocesses for dispensing drops to generate spotted arrays. The '193patent, and U.S. Pat. No. 5,885,837 to Winkler, also describe separatingreactive regions of a substrate from each other by inert regions andspotting on the reactive regions. The '193 and '837 patents are herebyincorporated by reference in their entireties. Other techniques arebased on ejecting jets of biological material to form spotted arrays.Other implementations of the jetting technique may use devices such assyringes or piezo electric pumps to propel the biological material.

Synthesized or spotted probe arrays typically are used in conjunctionwith tagged biological samples such as cells, proteins, genes or EST's,other DNA sequences, or other biological elements. These samples,referred to herein as “targets,” are processed so that they arespatially associated with certain probes in the probe array. Forexample, one or more chemically tagged biological samples, i.e., thetargets, are distributed over the probe array. Some targets hybridizewith at least partially complementary probes and remain at the probelocations, while non-hybridized targets are washed away. Thesehybridized targets, with their “tags” or “labels,” are thus spatiallyassociated with the targets' complementary probes. The hybridized probeand target may sometimes be referred to as a “probe-target pair.”Detection of these pairs by scanners can serve a variety of purposes,such as to determine whether a target nucleic acid has a nucleotidesequence identical to or different from a specific reference sequence.See, for example, U.S. Pat. No. 5,837,832, referred to and incorporatedabove. Other uses include gene expression monitoring and evaluation(see, e.g., U.S. Pat. No. 5,800,992 to Fodor, et al.; U.S. Pat. No.6,040,138 to Lockhart, et al.; and International App. No.PCT/US98/15151, published as WO99/05323, to Balaban, et al.), genotyping(U.S. Pat. No. 5,856,092 to Dale, et al.), or other detection of nucleicacids. The '992, '138, and '092 patents, and publication WO99/05323, areincorporated by reference herein in their entirety for all purposes forthe uses stated above and all the uses that are disclosed therein.

To ensure proper interpretation of the term “probe” as used herein, itis noted that contradictory conventions exist in the relevantliterature. The word “probe” is used elsewhere in some contexts to refernot to the biological material that is synthesized on a substrate ordeposited on a slide, as described above, but to what has been referredto herein as the “target.” To avoid confusion, the term “probe” is usedherein to refer to probes such as those synthesized according to theVLSIPS™ technology; the biological materials deposited or positioned soas to create spotted arrays; and materials synthesized, deposited, orpositioned to form arrays according to other current or futuretechnologies. Moreover, as noted, the term “probe” is not limited toprobes immobilized in array format. Rather, the functions and methodsdescribed herein may also be employed with respect to other parallelassay devices and techniques. Also, in some cases the sequence and/orcomposition of the probes may not be known, or may not be fully known.

Noise Compensation Module 310.

FIG. 3 is a functional block diagram of a scanner-computer systemshowing scanner 300 under the control of computer 350. A component ofscanner 300 is noise compensation module 310 that, among other things,conditions emission signals 192 and excitation signals 194 to providebidirectional edge and feature clarification. In the illustratedimplementations, this function is accomplished using an excitationsignal filter having appropriate smoothing characteristics andsymmetrical rise and fall characteristics, and a matched emission signalfilter. In some implementations, such as shown in FIG. 4, module 310also includes a hardware-implemented normalization factor generator 450.Generator 450 generates a normalization factor 452 that is provided tocompensation processor 460. Based on normalization factor 452, processor460 adjusts emission signals 192 for noise in excitation signals 194.The resulting normalized emission signal 312 is shown in FIG. 3 indotted lines to emphasize that it is the product of an optionalconfiguration. In some other embodiments, a software applicationexecuted on computer 350, together with a digital signal processor boardin the computer, implement the functions of generating a compensatingfactor and adjusting emission signals 192 to compensate for noise inexcitation signals 194. The result is normalized emission signal data374 stored in system memory 370 of computer 350 for further processingand/or display. The functions of certain components of scanner 300 andcomputer 350 are interchangeable in some implementations. As anon-limiting example, some or all of the functions of digital signalprocessor board 380, as well as those of scanner control and analysisapplications executables 372 may, in some implementations, be carriedout by noise compensation module 310. The functions of module 310 arenow further described, first in an example in which module 310 carriesout both bi-directional edge clarification and noise compensation, andthen in an example in which the latter function is carried out bysoftware executing on computer 350.

FIG. 4 is a functional block diagram of one implementation of noisecompensation module 310, referred to as module 310A. Using primarilyhardware components, illustrative module 310A adjusts emission signal192 to compensate for noise in excitation source 120, thereby providingnormalized emission signal 312. Module 310A includes a series of anumber N gain generators 410. Under the control of computer 350, gaingenerators 410 provide adjustable gains so that excitation signal 194 isnormalized irrespective of excitation source. This normalization isprovided to compensate for differences in the amplitude of excitationsignal 194 due to differences in optical parameters of scanner opticsand detectors 100 at different wavelengths of excitation beam 135. Theseoptical parameters include the percentage of light of differentwavelengths that passes through dichroic mirror 136, optical losses dueto mirror reflection that may vary according to wavelength, the responsecharacteristics of excitation detector 110 as a function of wavelength,and other factors that will be appreciated by those of ordinary skill inthe relevant art.

Gain generators 410 may be implemented in accordance with any of avariety of conventional techniques, or ones that may be developed in thefuture, for changing the amplitude of a signal. The gains provided byeach of generators 410 may be predetermined based on calibrationprotocols; for example, variable resistors may be adjusted by atechnician during manufacture so that a standard amount of powerprovided by each of excitation sources 120 results in a same voltage forgain-normalized excitation signal 422. Alternatively, this adjustmentfunction could be performed automatically by scanner control andanalysis executables 372. For example, under the control of executables372, each of excitation sources 120 may successively be enabled and arepresentative value of excitation signal 194 be calculated for each. Inaccordance with known techniques, these calculated values may beprovided to generators 410 to change gain parameters to achieve signalnormalization.

Module 310A also includes multiplexer 420 that, under the control ofcomputer 350 in this implementation, selects the normalized excitationsignal provided by the gain generator corresponding to the one ofexcitation sources 120 that is operational. For example, during a periodwhen computer 350 has made excitation source 120A operational, andassuming for illustrative purposes that gain generator 410A has beencalibrated to source 120A, then computer 350 provides an appropriateenabling or control signal to multiplexer 420 so that the signal fromgenerator 410A is selected. Multiplexer 420 may select from any numberof “N” inputs to provide, in the illustrated implementation, oneselected output, shown in FIG. 4 as gain-normalized excitation signal422. Any of numerous conventional or future multiplexers or switches maybe used to provide this function.

Another component of module 310A in the illustrated implementation isgain generator 490. Generator 490 adjusts the gain of emission signal192 in accordance with known techniques to provide that the input tofilter 440 is within a nominal range of amplitudes or, alternatively,has a nominal steady-state component. Thus, for example, if theemissions of a particular fluorophore in a particular assay occur over arelatively small dynamic range, the emission signal from thatfluorophore may optionally be adjusted by gain generator 490 to providea proportionately larger-range signal for filtering and subsequentsampling. The magnitude of the gain adjustment may, in someimplementations, be user selected. For example, a user may employ agraphical user interface (not shown) or other input technique to specifyto scanner control and analysis executables 372 what the gain providedby gain generator 490 should be. This determination typically is madebased on the fluorophores used in a particular assay and a list ofillustrative gains that may be presented to the user in a pull down menuof the graphical user interface or in accordance with any of a varietyof other known techniques. In other implementations, the gain value maybe determined automatically by scanner control and analysis executables372. For example, executables 372 may measure emission signal 192 todetermine its low-frequency components, peak-to-peak amplitudes, orother indicators of dynamic range. In accordance with known techniques,executables 372 may then consult a look-up table included, for example,in calibration data 376, to compare the measured indicators with nominalvalues, and adjusts the gain accordingly.

Also included in noise compensation module 310A is emission filter 440.Emission filter 440 performs an anti-aliasing function so thatnormalized emission signal 312 may be digitized without aliasing errors.In the illustrated implementation, filter 440 is a low-pass filter. Asnoted, it is not uncommon for high-frequency noise to be present in theoutputs of less expensive lasers, and the magnitude of this noise mayconstitute a substantial portion (e.g., 60%) of the magnitude of thesignal. Generally, emissions of fluorophores are linearly related totheir excitation throughout a range of interest in typical scannerapplications. Therefore, high frequency noise from the lasers inexcitation beam 135 produces high frequency noise of the same characterin emission signal 192 and adjusted emission signal 462 and, of course,in excitation signal 194.

The low-pass, anti-aliasing characteristics of filter 440 are designed,in accordance with known techniques, based on a rate at which normalizedemission signal 312 will be digitally sampled. This sampling rate, inturn, is based on a desired scan rate and a desired resolution of thescanned image. These considerations are now described in relation toFIGS. 5A and 5B.

FIG. 5A is a simplified graphical representation of a probe feature fordetection and processing by scanner 300. Generally, the term “probefeature” is used in this context to refer to a region of a probe arraymade up one or more probes that are designed to detect a same target orportion of a target, or to provide a control to verify the detection ofthat target or portion of a target. In a spotted probe array, a probefeature may be a single spot of a biological material intended tocontain one species of polymer. In a synthesized probe array, a probefeature may include many thousands of oligonucleotides designed for aperfect match with a same target sequence, or a probe feature mayinclude thousands of nucleotides designed for a mismatch (for controlpurposes) with a same target sequence.

FIG. 5A shows idealized probe feature 500 in the form of a singlecircular spot that may be deposited, for example, by an Affymetrix® 417™or 427™ Arrayer. It will be understood that a probe feature may be anyshape, including irregular shapes. Spot 500 may be, as an illustrativeexample, one of features 230 of the spotted probe array of FIGS. 2A and2B. In the manner described above, objective lens 145 scans over probefeature 500 (and, typically, other probe features of the probe array) inbi-directional arcs. An illustrative scan 520 is shown in FIG. 5A. Itwill be understood that FIG. 5A is not necessarily drawn to scale, andthat the ratio of the radius of the arc of scan 520 to the radius offeature 500 is illustrative only.

Also, probe feature 500 moves under objective lens 145, as representedby direction 244 of FIGS. 2B and 5A that, as noted, may be referred tofor convenience as the “y” direction. Thus, in the illustratedimplementation, arm 200 scans in an arc in one direction, shown asleft-to-right scan 520 in FIG. 5A. Translation stage 242 is then movedincrementally by a stepping motor (not shown) in y-direction 244 and arm200 then scans back in the opposite direction, shown as right-to-leftarcuate scan 522. Translation stage 242 is again moved in direction 244,and so on in scan-step-scan-step sequences. In this example, the terms“scan” or “scan line” thus will be understood to apply to a scan of aline or an arc, typically each “scan” referring to the movement alongthe line or arc in one direction. However, a scan may also be repeatedin one direction, or bi-directionally, multiple times, and the terms“scan” or “scan line” may refer to these repeated scans in somecontexts. Returning to the present specific example in which scans 520and 522 are referred to as separate scans, the distance between thesescans thus corresponds to the distance that translation stage 242 ismoved in each increment, although it will be understood that thedistance shown in FIG. 5A is not necessarily to scale and isillustrative only. It will be understood that any other combination ofscanning and stepping is possible in alternative implementations, andthat scanning and moving of translation stage 242 may occur at the sameor at overlapping times in some implementations. Translation stage 242need not be stepped in some implementations, but may, for example, bemoved continuously.

FIG. 5B is a plot having a pixel clock axis 530 showing when clockpulses 532 occur. Axis 530 in the illustrated implementation is aspatial axis; that is, each of clock pulses 532 occurs in reference tothe radial location of arm 200 during each scan, as described in greaterdetail below. Thus, with reference to the position of translation stage242 indicated by scan 520, a clock pulse 532A occurs prior to arm 200passing over feature 500 from the left as shown in FIGS. 5A and 5B. (Forsake of clarity of illustration only, vertical dotted lines are providedbetween FIGS. 5A and 5B to illustrate this alignment.) As anotherexample, clock pulse 532C occurs with respect to scan 520 when arm 200has just passed over portions of feature 500 indicated by pixel areas Aand K. These areas are referred to as pixel areas because a digitalvalue is assigned to each such area in the illustrated implementationbased on the strength of a filtered emission signal associated with thatarea. In accordance with known techniques, clock pulses 532 enable thedigital sampling of the filtered emission signal.

As will be appreciated by those of ordinary skill in the relevant art,the Nyquist criterion may be applied to determine the appropriatelow-pass characteristics of filter 430 based on a desired sampling rate.As noted, clock pulses 532 are spatially rather than temporallydetermined in the illustrated implementation. Moreover, in some aspectsof the illustrated implementation, galvanometer 216 is driven by acontrol signal provided by computer 350 such that the velocity of arm200 in x-direction 243 is constant in time during those times when arm200 is over probe feature 500 (and, typically, over other features ofthe probe array being scanned). That is, dx/dt is a constant (and thusthe angular velocity varies) over the probe-scanning portions of eacharc and, in particular, it is a constant during the times when clockpulses are generated to enable digital sampling. As is evident, dx/dtmust be reduced to zero between each successive scan, but thisdeceleration and reversal of direction takes place after arm 200 haspassed over the probe feature (or, more generally, the probe array). Thedesign and implementation of a galvanometer control signal to provideconstant dx/dt are readily accomplished by those of ordinary skill inthe relevant art.

Thus, the approximate sampling rate may readily be calculated based onthe desired scanning speed (dx/dt) and desired pixel resolution. Toprovide an illustrative example, a spot deposited by an Affymetrix® 417™Arrayer typically has a diameter of approximately 200 microns. Spottedprobe arrays made using this instrument typically may be deposited overa surface having a width of about 22 millimeters on a microscope slidethat is 25 millimeters wide. In order to achieve pixel resolution ofabout 10 microns, a sampling rate of about 160 kHz is sufficient forscanning speeds typical for scanners used with respect to these probearrays, such as the Affymetrix® 428™ scanner. Other sampling rates,readily determined by those of ordinary skill, may be used in otherapplications in which, for example, different scanning speeds are usedand/or different pixel resolutions are desired. The desired pixelresolution typically is a function of the size of the probe features,the possibility of variation in detected fluorescence within a probefeature, and other factors. The desired scanning speed typically is afunction of the size of the probe array to be scanned, the amount of atime that a user may wish to wait for the scanning to be completed, theresponse characteristics of the fluorophores, the responsecharacteristics of emission detector 115, the response and operationalcharacteristics of galvanometer 216, and a variety of other factors.

In order to avoid aliasing errors, filter 440 should have a low-passcutoff frequency of one half or less of the sampling frequency, as thoseof ordinary skill will appreciate based on the Nyquist criterion. Thus,for example, filter 440 as implemented in the Affymetrix® 428™ scanneris designed in accordance with known techniques to have a cut-offfrequency of 33 kHz in some implementations and 67 kHz in otherimplementations. As will be evident to those of ordinary skill in therelevant art, the lower cut-off frequency achieves somewhat greatersmoothing at the expense of a potential loss in signal accuracy. Inimplementations in which the command signal driving galvanometer 216 isnot designed to provide constant dx/dt but rather, for example, aconstant angular velocity over the probe-scanning area, the appropriatecut-off frequency dictated by the Nyquist criterion should take intoaccount variation in the sampling rate for different portions of the arcassuming that it is desired to provide clock pulses that are constant inthe x direction.

Noise compensation module 310A also includes excitation signal filter430 that has the same design characteristics as, i.e., it is matchedwith, emission signal filter 440. The reason for matching filters 430and 440 with each other is to provide that the delay through bothfilters is the same. If the delays were different, then filteredexcitation signal 432 and filtered emission signal 442 would no longerbe spatially correlated. That is, a value of filtered excitation signal432 at a particular time “t” would represent the excitation of aparticular fluorophore at a position “p,” but the value of filteredemission signal 442 at the same time “t” would represent the emission ofa fluorophore that was excited at a position either before or afterposition “p” in the scanning arc.

Loss of spatial correlation could interfere with techniques describedherein to normalize emission signals to compensate for noise in laserexcitation signals. As described below, normalized emission signal 312is determined in this implementation by adjusting filtered emissionsignal 442 by a normalization factor 452. Factor 452 is determined bycomparing a nominal excitation value with filtered excitation signal432. The nominal excitation value can be derived in a variety of ways,such as by low-pass filtering or by taking an average or otherstatistical measure of large numbers of samples over a relatively longperiod so that the impact of noise components is minimized. Also, anominal value can be predetermined by manual calibration or othertechniques, and the value stored in calibration data 376 for reference.In essence, emission signals are adjusted to compensate for variationsin the excitation signals that gave rise to them. If spatial correlationis not maintained, then this cause and effect relationship may be lostand erroneous adjustments may result. However, approaches other thanmatched filters may be taken to provide spatial correlation. Forexample, in alternative embodiments, any mismatches in the delays offilters 430 and 440 may be compensated for either in hardware (e.g., byintroducing a compensating delay with respect to one or the other signalin accordance with known techniques) or in software (e.g., by realigningsampled emission and excitation signals to offset delays).

FIGS. 5A–5D further illustrate these points. In these figures, it isillustratively assumed that executables 372 initiates clock pulse 532Dat a time “t.” This clock event is determined by the receipt of a signalfrom transducer 215 indicating that excitation beam 135 is located overa radial position in the scanning arc shown with reference to scan 520as radial position 525C′. (Transducer 215 provides a signal with thesame value when beam 135 is located over the same radial position forscan 522, which is labeled as radial position 525C″.) Executables 372retrieves from galvo position data table 378 a value of the signal fromtransducer 215 that corresponds to radial positions 525C′ and 525C″.This value also corresponds to a position on each of every other scanthat intersects with constant radial position line 525C of FIG. 5A,which is parallel to direction 244 of translation stage 242.) Thisretrieved value is compared to the signal from transducer 215 until itis determined that the values are equal, causing executables 372 toinitiate clock pulse 532D. In the illustrated implementation, the radialposition values in galvo position data table 378 are predetermined sothat each of constant radial position lines 525 (e.g., 525A, 525B,through 525K of the example of FIG. 5A) are positioned at an equaldistance perpendicular to direction 244. This feature facilitates thesoftware translation of pixels from polar coordinates to Cartesiancoordinates.

Thus, returning to the example of clock pulse 532D initiated at time“t,” it is illustratively assumed that filtered excitation signal 432has a value at that time that is shown in FIG. 5D as filtered excitationvoltage 580D. Voltage 580D therefore may be spatially correlated withradial position 525C′ (and with the position at time “t” of translationstage 242). However, because voltage 580D has been filtered and thusphase delayed, it typically corresponds with values of gain-normalizedexcitation signal 422 that occurred earlier in time and therefore at alocation on scan 520 to the left of radial position 525C′. To take intoaccount this spatial translation, it is provided that adjusted emissionsignal 462 is phase delayed by the same amount. As noted, this result isaccomplished by providing that the phase delay characteristics offilters 440 and 430 are matched. For example, the same electricalcomponents may be used, particularly if gain generator 490 provides thatadjusted emission signal 462 has approximately the same dynamic range asgain-normalized excitation signal 422. Thus, because the phase delaysfor both excitation and emission signals are the same, the value offiltered emission signal 442 sampled as the result of the initiation ofclock pulse 532D, shown as illustrative voltage 550D in FIG. 5C,corresponds spatially with excitation voltage 580D. Alternativelystated, the matched filters provide that a fluorophore located at aposition on probe feature 500 represented by position 525C′, which wasexcited by voltage 580D, emitted a signal represented by voltage 550D,wherein both voltages were sampled at a same time.

A further desirable characteristic to be considered in the design offilters 430 and 440 is to provide constant group delay of the filters'input signals irrespective of the frequency components of those inputs.That is, it generally is desirable that the phase delay introduced byfilters 430 and 440 be a determinable constant based on the filters'design rather than the characteristics of the input signal.Alternatively stated, it generally is desirable for bi-directionalscanning that the rise and fall response characteristics of each of thefilters be symmetrical. In the illustrated implementation, thesecharacteristics are accomplished by providing that both of filters 430and 440 are linear-phase filters, such as Bessel filters. In particular,filters 430 and 440 are high-order Bessel filters, such as 6^(th) orderor higher, and preferably 11^(th) order or higher, Bessel filters. Theadvantage of providing the feature of symmetrical, matched filters maybe illustrated with respect to the example of FIGS. 6A–6E.

FIG. 6A shows a simplified input waveform 600 such as may be provided asinput to filter 440, i.e., adjusted emission signal 462. Input waveform600 has simulated high-frequency noise components that are includedwithin repeating dual pulses 610A through 610G (generally andcollectively referred to hereafter for convenience as noise pulses 610).It will be understood, however, that noise components of signal 462 (andof signal 422) typically are not regular and are more complex infrequency and amplitude.

Output waveform 615 of FIG. 6B is a graphical representation of theoutput of filter 440 in response to input waveform 600, assuming thatfilter 440 is not a linear-phase filter. For example, filter 440 may bea Butterworth filter in this example. A peak 612A is observable onwaveform 442A having a rising edge 616A that is steeper and of adifferent shape than its falling edge 614A; i.e., the rising and fallingedges are not symmetrical. As is evident, the same asymmetry occurs inresponse to each of input pulses 610, as indicated by output pulses 612Athrough 612H.

It is now illustratively assumed that output waveform 615 is the resultof scan 520 in the left-to-right direction, and that a successive scan522 in the opposite direction is made over a probe feature that has aconstant concentration of fluorophores between and including the twoscans. Equivalently, it may be assumed that the stepping motor does notadvance translation stage 242 between the scans. To obtain the sameresult irrespective of the direction of the scan, output waveform 615should have the same shape irrespective of which direction the scan wastaken (although the waveforms will be displaced in time by a factor oftwice the phase delay of the filter). However, as can be seen from FIG.6B, this desired symmetry will not occur since the rising and fallingedges of pulses 612 are asymmetrical. Improved smoothing and perhapsgreater symmetry can be accomplished by higher-order filters that do nothave the characteristic of constant group delay, as shown by FIG. 6C.Output waveform 620 of FIG. 6C is the response of such a filter to inputwaveform 600. Pulses 622 of FIG. 6C are smoother and somewhat moresymmetrical than pulses 612 of FIG. 6B, but the problem ofdirectional-dependent inconsistency remains.

In contrast, output waveform 630 of FIG. 6D is the simulated response offilter 440 implemented as a Bessel filter, and it may be observed thatthe resulting pulses are substantially symmetrical. For example, risingedge 636A is substantially the mirror image of falling edge 634H. Thedegree of symmetry generally may be improved by implementing filter 440(and providing matched filter 430) as higher-order Bessel filters, asshown by output waveform 640 of FIG. 6E.

The advantages of using symmetrical filters for bi-directional scanningmay further be appreciated with reference to FIGS. 7A–7C and 8A–8C. FIG.7A is a graphical representation of an input waveform 700 that isillustratively assumed to be provided to asymmetrical emission signalfilter 440. Waveform 800 of FIG. 8A is the same as waveform 700, exceptthat it is illustratively assumed to be provided to filter 440implemented as a symmetrical filter, such as Bessel filters. Bothwaveforms 700 and 800 are idealized for clarity of illustration in thatno noise is present and the pulses are shown as simple step functions.

FIG. 7B shows output waveform 739 resulting from a scan 720 in a firstdirection, and FIG. 7C shows output waveform 749 resulting from a scan522 in an opposite direction, both in the context of an implementationof filter 440 (and thus filter 430 to provide matching) as asymmetricalfilters. Similarly, FIG. 8B shows output waveform 839 resulting from ascan 820 in a first direction, and FIG. 8C shows output waveform 849resulting from a scan 522 in an opposite direction, both in the contextof an implementation of filters 430 and 440 as symmetrical filters. Thevoltage waveforms of FIGS. 7A–7C and 8A–8C are shown as functions ofconsecutive clock pulses 732 and 832, respectively, on spatial pixelclock axes. With reference to FIG. 7B, output waveform 739 results fromscanning in left-to-right scan direction 720 in relation to spatialpixel clock axis 730. That is, input waveform 700 results from scanningat positions to the left of the radial position represented by pixelclock position 732D and proceeding toward the radial positionrepresented by pixel clock position 732K. As seen from output waveform739, the portion of the rising edge of that waveform between positions732D and 732E is of a different shape than the portion of the fallingedge between positions 732F and 732G. This difference is graphicallyrepresented by pixels 735 aligned below corresponding pixel clockpositions. For example, if waveform 739 were sampled by a pixel clockpulse corresponding to position 732D, the sampled value would berelatively small. For convenience, a small value (e.g., relatively lowintensity of filtered emission signal 442) is shown as a dark pixel,such as pixel 735A. If waveform 739 were sampled at position 732E, atthe top of the rising edge, the sampled value would be relatively large,as represented by white pixel 735B indicating a high intensity emissionsignal. A sample taken at position 732F along the falling edge providesa middle value, as represented by pixel 735C in cross hatching. The areascanned in direction 720 between positions 732D and 732K would berepresented by the following sequence of pixel intensities as indicatedby pixels 735: dark—white—mid—dark—dark—white—mid—dark. Each level ofintensity is indicative of a different degree of emission byfluorophores at locations corresponding to the pixel clock positions(although, as noted, a phase delay is anticipated). In FIG. 7C it isillustratively assumed that the same input waveform 700 is provided tothe same filter, except that the scan is in direction 722, i.e.,starting from position 732K and moving toward position 732D. Because ofthe asymmetry of the rising and falling edges after filtering, thesamples taken in this reverse positional order result in a differentsequence of pixel intensities, as indicated by pixels 745:dark—mid—white—dark—dark—mid—white—dark.

Two observations can thus be made by comparing these two sequences. Oneobservation is that the two pulses of input waveform 700 are measureddifferently based on the direction of scanning: in the 720 direction thepulses are measured as white—mid, whereas in the 722 direction the samepulses are measured as mid—white. That is, in successive bid-directionalscans, spatial transitions in input values provide inconsistent samplingresults. The second observation is that the measured pulses are shiftedspatially by twice the phase delay of the filter. This phase delay neednot, and generally will not, be an integer number of pixel positions,but is a real number representing a spatial shift determined by thephase delay characteristics of the filter.

The results of these effects can be seen in FIG. 7D, which is asimplified graphical representation of pixels resulting from successivebidirectional scans of a probe feature in which adjusted emission signal462 is represented by input waveform 700 for all scans in bothdirections. The pixels are aligned spatially along pixel clock axis 730,as in FIGS. 7B and 7C. In order to accommodate the phase delayintroduced by emission filter 440, the spatial width of the scan ineither direction (a distance D equal to the distance between ten clockpulses in this example) is greater than the spatial width of the probefeature being scanned (eight clock pulses); i.e., the feature can besaid to have been “over-scanned” in both directions. Although the phasedelay in this example is shown as equal to the distance betweensuccessive clock pulses, this is only an illustrative example. As noted,the delay in general is measured as a real number that may be expressedas fractions (any real number) of distances between clock pulses, i.e.,fractional pixels.

FIG. 7E is a graphical representation of the pixels of FIG. 7D shiftedso as to graphically compensate for the effects of phase delay. Thepixels at the beginning and end of each scan have been eliminated fromthis representation so that the external edges of the featurerepresentation can be more clearly seen. This shifted representationmay, for example, be presented to users in a graphical user interface,or the data associating the pixels in this manner may be stored incomputer 350 for further image and/or signal processing. These tasks maybe accomplished in accordance with any of a variety of conventionaltechniques well known to those of ordinary skill in the art whetherimplemented in hardware, software, or a combination thereof. Asindicated by FIG. 7E, transient pixel intensities appear indistinct. Inparticular, the left hand edge of the feature as represented by inputwaveform 700, as detected at the output of asymmetric filter 440 of thisexample, has a zipper-like pattern of alternating high andmid-intensities shown by vertically alternating pixels 735B and 745B.The right hand edge has the same distorted quality, as shown byvertically alternating pixels 735G and 745G. The same distortion ispresent at transient points within the feature, as indicated byvertically alternating pixels 735C and 745C, and by verticallyalternating pixels 735F and 745F.

FIGS. 8D and 8E present the same information as described above withregard to FIGS. 7D and 7E, respectively, except that FIGS. 8D and 8E arebased on the assumption of FIGS. 8B and 8C that filter 440 is asymmetrical filter. As is indicated by FIGS. 8D and 8E, a phase delay isalso introduced by the symmetric filter. However, transients in theintensity of filtered emission signal 442 are not distorted. Rather, asindicated by the vertical alignment of consistently high-intensitypixels, the zipper effect is avoided and the transient points are clear.

Returning now to FIG. 4, two additional components of noise compensationmodule 310A remain to be described. Normalization factor generator 450compares filtered excitation signal 432 to excitation reference 476. Thepurpose is to provide a normalization factor 452 that provides a measureof a deviation of filtered excitation signal 432 from a nominal value.This deviation typically is due to noise in excitation beam 135generated by excitation source 120 (e.g., the laser operational at theperiod of interest) that thus appears in excitation signal 194 andgain-normalized excitation signal 422 and, in filtered form, in filteredexcitation signal 432. The deviation may also be due, in someimplementations, to long-term drift in excitation beam 135. For example,lasers may degrade over periods of weeks, months, or years so that themagnitude of their steady state output declines. This long-term drift,which may be positive or negative, may be compensated for by adjustingthe gain of the appropriate one of gain generators 410B. Alternatively,the excitation reference component of calibration data 376 may beadjusted accordingly. In either case, if the long-term drift reaches athreshold level, a user may be advised that excitation source 120 hasdegraded and should be repaired or replaced. The threshold level may,for example, be a predetermined percentage of a nominal laser outputvalue supplied by the laser manufacturer and stored as a component ofcalibration data 376. The notification to the user may be accomplishedfor example by an appropriate graphical user interface of computer 350,all in accordance with conventional techniques well known to those ofordinary skill in the relevant art.

With respect to the objective of compensating for laser noise,excitation reference 476 typically, but not necessarily, represents anominal expected value of filtered excitation signal 432 over a timeperiod substantially greater than that of the lowest expected noisefrequency, but shorter than the time over which significant laser drifttypically occurs. This period is selected such that an average or otherstatistical measure of signal 432 may reliably be determined. Forexample, monitored filtered excitation signal 434, equal to orrepresentative of filtered excitation signal 432, may be provided tocomputer 350 for digital sampling over a period of one or more scans. Inaccordance with known techniques, the sampled signal may bestatistically processed to provide, for example, an average or nominalvalue of signal 432 for that scan that may be used as excitationreference 476 for the next scan. Alternatively, each scan may be donetwice: once to determine an average value and once to determine actualvalues including noise. Thus, the value of excitation reference 476 maybe updated as frequently as every scan or less. Alternatively, reference476 may be predetermined based on an initial calibration of excitationsource 120, or based on manufacturer's specifications, and stored as acomponent of calibration data 376. In yet another alternativeimplementation, low-pass filter 431 may be used to remove all expectednoise components from filtered excitation signal 432, and thislow-frequency signal, shown as 476′, may be used as an excitationreference. This implementation is represented in dashed lines in FIG. 4to indicate its optional application.

As will now be appreciated by those of ordinary skill in the art, manyother techniques are possible in hardware, software, or both, forproviding an excitation reference that represents filtered excitationsignal 432 with noise components substantially removed. In many cases,moreover, it is desirable to provide that, at a standard value of gaingenerator 490, each implementation of each excitation source 120 of eachscanner 300 provides a standard value of filtered emission signal 442when exciting a standard fluorophore sample. In some implementations,therefore, a known concentration of fluorophores is prepared as acalibration slide to be scanned by each of a series of manufacturedscanners 300. Gain generator 490 is set at a nominal, standard, value,preferably one determined with respect to the dynamic range of thefluorophore. A photomultiplier tube or other detector is set to measurefiltered emission signal 442. In some implementations, adjusted emissionsignal 462 may be measured instead. The power of excitation beam 135 isalso measured by, for example, measuring filtered excitation signal 432(with the corresponding gain generator 410 set to a standard value) inaccordance with any of a variety of conventional measuring techniques.For each of excitation sources 120, e.g., for each of lasers 120A and120B of the illustrated implementation, the excitation source isadjusted to increase or decrease excitation beam 135 until the measuredvalue of filtered emission signal 442 (or of signal 462) is a standardvalue. The value of filtered excitation signal 432 at this calibrationsetting is stored in calibration data 376 and thenceforth serves asexcitation reference 476 for that excitation source 120 for that scanner130, or as a basis for determining an appropriate excitation reference.In alternative implementations, instead of adjusting the excitationsource to increase or decrease beam 135, the adjustment may be made togain generator 490, and/or to the gain of emission detector 115. Ineither case, excitation reference 476 is a constant value, and is notdetermined by, for example, averaging scans or low-pass filtering asdescribed above. Rather, reference 476 is a calibrated value unique tothe instrument and, generally, constant for the life of the instrumentor a period of time over which consistent experimental results aredesired. A significant advantage of the calibration approach leading toa constant excitation reference 476 for each scanner 130 instrument isthat, even if excitation source 120 degrades over time, a user will beable to replicate experiments and obtain the same measurements overtime.

In one implementation, normalization factor generator 450 may be ananalog multiplier/divider device, such as is available from a variety ofcommercial suppliers including Analog Devices, Inc. of Norwood, Mass.,or one of numerous other analog and/or digital devices that performmultiplication, division, and/or comparative functions. In theillustrated implementation, generator 450 multiplies or divides filteredexcitation signal 432 and excitation reference 476 (or 476′) to providea ratio between the two. For example, if reference 476 has a value of1.00 and the value of signal 432 is 1.25, then the value ofnormalization factor 452 at that time (hereafter, for convenience, the“instantaneous” value) is 1.00/1.25=0.80. That is, in this illustrativeimplementation, the comparison between a steady state measure of signal432 and the instantaneous value of signal 432 is provided by dividing anaverage value of signal 430 by the instantaneous value of signal 430.Any of various other statistical comparisons may be used in alternativeimplementations, as will now be appreciated by those of ordinary skillin the relevant art.

Comparison processor 460 applies normalization factor 452 to filteredemission signal 442. Comparison processor 460 may also any of numerousanalog multiplier/divider device or other devices such as noted abovewith respect to generator 450. In the illustrated implementation,processor 460 multiplies the instantaneous value of filtered emissionsignal 442 by normalization factor 452 to provide normalized emissionsignal 312. Thus, in the illustrated implementation, generator 450 andprocessor 460 together implement a function that may be represented as:signal 312(t)=signal 442(t)*(average signal 432/signal 432(t))where (t) indicates instantaneous values over time.

Signal 312 may in some implementations be provided to computer 350 forsampling as enabled by pixel clock pulses generated by computer 350, asdescribed above. In other implementations, the generation of pixel clockpulses and their application to signal 312 may be done by an appropriatedevice located in scanner 300. For example, as shown in FIG. 3, pixelclock 305 may be implemented in scanner 300 by a complex programmablelogic device (CPLD) such as is commercially available from AlteraCorporation of San Jose, Calif., and other suppliers.

FIG. 9 is a functional block diagram showing an alternativeimplementation in hardware and software of the functions just describedwith respect to the primarily hardware implementation of FIG. 4. Inparticular, the functions of elements 910, 920, 930, 940, and 960 ofFIG. 9 are similar to those described above with respect to elements410, 420, 430, 440, and 460 of FIG. 4, respectively. However, in theimplementation of FIG. 9, the functions described above with respect tonormalization factor generator 450 and comparison processor 460 areperformed by computer 350. The result in the implementation of FIG. 9 isnormalized emission signal data 374 that provide information similar tothat contained in normalized emission signal 312 as shown in FIG. 4.

It will be understood that the embodiment of computer 350 shown in FIGS.3, 4 and 9 is exemplary only, and that many alternative implementationsare possible. For example, the functions of computer 350 may beperformed by one or more components of scanner 300 rather than beingperformed by an external computer. For instance, scanner 300 may includea microprocessor with associated firmware for performing some or all ofthe functions ascribed herein to computer 350.

In the illustrated embodiment, computer 350 may be located locally toscanner 300, or it may be coupled to scanner 300 over a local-area,wide-area, or other network, including an intranet and/or the Internet.Computer 350 may be a personal computer, a workstation, a server, or anyother type of computing platform now available or that may be developedin the future. As shown in FIG. 4, computer 350 includes a processcontroller 462 for performing control and analysis functions withrespect to scanner 300 as described below. Typically, computer 350 alsoincludes known components such as CPU 355, operating system 360, systemmemory 370, memory storage devices 380, and input-output controllers375, all of which typically communicate in accordance with knowntechniques such as via system bus 390. In the illustratedimplementation, computer 350 also includes digital signal processorboard 380, which may be any of a variety of PC-based DSP controllerboards, such as the M44 DSP Board made by Innovative Integration of SimiValley, Calif. A variety of other components may be included in computer350, as is well known by those of ordinary skill in the relevant art.

In reference to FIG. 9, filtered excitation signal 932, corresponding tofiltered excitation signal 432 described above, is provided to linedriver 931 that, in accordance with known techniques, provides signal932′ to DSP board 380 of computer 350. Similarly, filtered emissionsignal 942, corresponding to filtered emission signal 442 describedabove, is provided to line driver 941 that provides signal 942′ to DSPboard 380. In accordance with known analog-to-digital samplingtechniques, board 380 samples signals 932′ and 942′ based on pixel clocksampling pulses. These pulses may be provided by pixel clock CLPD 305,or they may be generated by aspects of executables 372 by comparingradial position information from galvo position transducer 215 with datain galvo position data table 378. In the former implementations, CLPD305 performs the functions of computer 350, and may also store data suchas that in table 378. Optionally, prior to sampling, board 380 mayinclude anti-aliasing filters (such as filters 1030 and 1040 of FIG. 10)designed in accordance with the Nyquist criterion as noted above tofurther ensure that aliasing errors do not occur when signals 932′ and942′ are sampled.

In this alternative implementation software-implemented functionalelements of executables 372 perform the functions described in referenceto FIG. 4 with respect to the operations of normalization factorgenerator 450 and comparison processor 460. That is, executables 372, incooperation with other elements of computer 350 such as CPU 355 andoperating system 380, generates a compensation factor that is based on acomparison between a reference excitation value and, in someimplementations, each sampled excitation value. Executables 372 thenapplies this factor to each corresponding sampled emission signal toprovide normalized emission signal data corresponding to each sample ofthe emission signal. Thus, the functional elements of executables 372comprise sets of software instructions that cause the describedfunctions to be performed. These software instructions may be programmedin any programming language, such as C++ or another high-levelprogramming language. Executables 372 may therefore be referred to as “aset of scanner control and analyzing instructions,” and its functionalelements may similarly be described, for example, as sets ofnormalization factor generating instructions (as represented bygenerator 1050) and comparison processing instructions (as representedby processor 1060).

These operations are shown in greater detail in FIG. 10, which is afunctional block diagram of aspects of computer 350 that generatenormalization factors for each sampled excitation signal and appliesthose factors to filtered emission signals to obtain normalized emissionsignal data. In accordance with any of a variety of known techniques,analog to digital converter 1035 digitizes samples of signals 932′ togenerate excitation samples 1037, which are provided by to normalizationfactor generator 1050 of executables 372. Similarly, analog to digitalconverter 1045 digitizes samples of signals 942′ to generate emissionsamples 1047, which are provided by to comparison processor 1060 ofexecutables 372. For ease of reference, a pair of samples 1037 and 1047sampled according to the same sampling pulse will be referred to as aparticular instance of those samples.

Excitation reference 1076 of calibration data 376 also is provided togenerator 1050. Reference 1076 is a reference excitation value derivedin accordance with any of the techniques described above with respect toexcitation reference 476.

Generator 1050 performs functions similar to those described above withrespect to generator 450. For example, in some implementations,generator 1050 determines an instance of compensation factor 1052 bydividing reference 1076 by the value of excitation sample 1037 for thatinstance. This instance of factor 1052 is multiplied by thecorresponding instance of emission sample 1047 to obtain thecorresponding instance of normalized emission signal data 374. Thus, inthe illustrated and non-limiting implementation, each instance (I) ofdata 374 is derived in accordance with the algorithm:data 374(I)=sample 1047(I)*(reference 1076/sample 1037(I))

Typically, generator 1060 and processor 1070 are implemented as softwareinstructions in any appropriate programming language, such as C++, andcompiled for inclusion in executables 372 that are executed on computer350 of the illustrated implementation. In particular, system memory 370of computer 350 may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage devices 380 may be any of a variety ofknown or future devices, including a compact disk drive, a tape drive, aremovable or internal hard disk drive, or a diskette drive. Such typesof memory storage devices 380 typically read from, and/or write to, aprogram storage medium (not shown) such as, respectively, a compactdisk, magnetic tape, removable or internal hard disk, or floppydiskette. Any of these program storage media, or others now in use orthat may later be developed, may be considered a computer programproduct. As will be appreciated, these program storage media typicallystore a computer software program and/or data. Computer softwareprograms, also called computer control logic, typically are stored insystem memory 370 and/or the program storage medium used in conjunctionwith memory storage devices 380.

In some implementations, a computer program product is describedcomprising a computer usable medium having control logic (computersoftware program, including program code) stored therein. The controllogic, when executed by processor 355, causes processor 355 to performthe functions of scanner control and analysis executables 372, includinggenerator 1050 and processor 1060. In other embodiments, these and otherfunctions of executables 372 may be implemented primarily in hardwareusing, for example, a hardware state machine. Implementation of thehardware state machine so as to perform the functions of executables 372described herein will be apparent to those skilled in the relevant art.

Having described various embodiments and implementations, it should beapparent to those skilled in the relevant art that the foregoing isillustrative only and not limiting, having been presented by way ofexample only. Many other schemes for distributing functions among thevarious functional elements of the illustrated embodiment are possible.The functions of any element may be carried out in various ways inalternative embodiments.

Also, the functions of several elements may, in alternative embodiments,be carried out by fewer, or a single, element. For example, excitationsignal filter 430 and emission signal filter 440 of the implementationshown in FIG. 4 may, in other implementations, be replaced by a singleanti-aliasing filter that operates on the output of comparison processor460 to provide normalized emission signal 312. As another example, thefunctions of gain generators 410 could alternatively be performed bymultiplexer 420, or by computer 350.

Similarly, in some embodiments, any functional element may performfewer, or different, operations than those described with respect to theillustrated embodiment. Also, functional elements shown as distinct forpurposes of illustration may be incorporated within other functionalelements in a particular implementation. Furthermore, the sequencing offunctions, or portions of functions, generally may be altered. Forinstance, the functions of gain generator 490 may be performed afterthose of emission signal filter 440.

Certain functional elements, files, data structures, and so on, aredescribed in the illustrated embodiments as located in system memory 370of computer 350. In other embodiments, however, any or all of these maybe located on, or distributed across, computer systems or otherplatforms that are co-located and/or remote from each other. Inaddition, it will be understood by those skilled in the relevant artthat control and data flows between and among functional elements andvarious data structures may vary in many ways from the control and dataflows described above. More particularly, intermediary functionalelements may direct control or data flows, and the functions of variouselements may be combined, divided, or otherwise rearranged to allowparallel processing or for other reasons. Also, intermediate datastructures or files may be used and various described data structures orfiles may be combined or otherwise arranged. Numerous other embodiments,and modifications thereof, are contemplated as falling within the scopeof the present invention as defined by appended claims and equivalentsthereto.

1. A method for analyzing molecules, comprising the steps of: (1)directing at least one filtered excitation beam from at least oneexcitation source to a first substrate having a control; (2) determiningone or more excitation values which related to the at least onefiltered-excitation beam directed to the control; (3) detecting anemission signal having one or more emission values; (4) adjusting the atleast one excitation source to obtain an excitation reference value whenthe one or more emission values match a standard value from the control;(5) scanning a plurality of pixel locations on a second substrate havinga plurality of probe locations using the excitation reference value togenerate a normalization factor; (6) adjusting at least one of the oneor more emission values based, at least in part, on the normalizationfactor; and (7) generating one of either a normalized emission signaland a normalized emission signal data in response to said adjusting ofsaid at least one of the one or more emission values.
 2. A methodcomprising the steps of: (1) scanning a plurality of pixel locations ona substrate having a plurality of probe locations to determine anaverage excitation value; (2) updating an excitation reference valuefrom the average excitation value; (3) directing an excitation beam tothe plurality of pixel locations on the substrate; (4) determining oneor more representative excitation values, each related to a value of theexcitation beam as directed to at least one of the plurality of pixellocations; (5) detecting an emission signal having one or more emissionvalues; (6) correlating each of the one or more emission values witheach of the one or more representative excitation values; (7) comparingthe excitation reference value to at least one of the one or morerepresentative excitation values, thereby generating a normalizationfactor; (8) adjusting at least one of the one or more emission valuesbased, at least in part, on the normalization factor; and (9) generatingone of either a normalized emission signal and a normalized emissionsignal data in response to said adjusting of said at least one of theone or more emission values.
 3. The method of claim 2, wherein: one ormore probes of a biological microarray are disposed in relation to thesubstrate.
 4. The method of claim 3, wherein: the one or more probes aredisposed at different probe locations on a surface of the substrate. 5.The method of claim 2, wherein: the substrate comprises a plurality ofdifferent polymer sequences coupled to a surface of the substrate. 6.The method of claim 5, wherein: the plurality of different polymersequences comprises a plurality of different oligonucleotides sequences,wherein each of the different polymer sequences is coupled in adifferent probe location of the surface.
 7. The method of claim 6,wherein: each of the probe locations has an area of one-hundredth of asquare centimeter or less.
 8. The method of claim 2, wherein: thesubstrate comprises more than one thousand different ligands of knownsequence collectively occupying an area of less than one squarecentimeter, the different ligands occupying different known locationswithin the area.
 9. The method of claim 2, wherein: the substrate has asurface comprising more than ten different nucleic acids of knownsequences at a plurality of probe locations on the surface of thesubstrate, each of the probe locations having an area of one-hundredthof a square centimeter or less, at least one of the nucleic acids beingbound to a labeled receptor.
 10. The method of claim 2, wherein: theexcitation beam is a laser beam.
 11. The method of claim 2, wherein: theplurality of pixel locations comprises one or more scan lines.
 12. Themethod of claim 2, wherein: the representative excitation values areeach related to a power of the excitation beam.
 13. The method of claim2, wherein: step (2) includes the steps of (a) directing the excitationbeam to a dichroic mirror, and (b) determining the representativeexcitation values based on a partial excitation beam that passes throughthe dichroic mirror.
 14. The method of claim 2, wherein: the emissionsignal arises from the direction of the excitation beam to the pluralityof pixel locations.
 15. The method of claim 14, wherein: the emissionsignal comprises a fluorescent signal.
 16. The method of claim 15,wherein: the substrate has a surface comprising a plurality of differentprobes at a plurality of probe locations on the surface of thesubstrate, at least one of the probes at a first probe location beingbound to a fluorescently labeled receptor, wherein at least one of theemission values corresponds to an emission from the fluorescentlylabeled receptor responsive to the excitation beam being directed to thefirst probe location.
 17. The method of claim 16, wherein: the probescomprise a plurality of different nucleic acids of known sequences. 18.The method of claim 2, wherein: step (4) includes spatially correlatingthe emission values with the representative excitation values.
 19. Themethod of claim 18, wherein: step (4) includes providing that eachemission value is correlated with at least one representative excitationvalue that is related to a value of the excitation beam that gave riseto the emission value.
 20. The method of claim 18, wherein: step (2)includes determining a first representative excitation value related toa power of the excitation beam as directed to a first of the pluralityof pixel locations; step (3) includes detecting an emission valuearising from the direction of the excitation beam to the first pixellocation; and step (4) includes correlating the first emission valuewith the first representative excitation value.
 21. The method of claim1, wherein: one or more probes of a biological microarray are disposedin relation to the second substrate.
 22. The method of claim 21,wherein: the one or more probes are disposed at different probelocations on a surface of the second substrate.
 23. The method of claim1, wherein: the second substrate comprises a plurality of differentpolymer sequences coupled to a surface of the second substrate.
 24. Themethod of claim 23, wherein: the plurality of different polymersequences comprises a plurality of different oligonucleotides sequences,wherein each of the different polymer sequences is coupled in adifferent probe location of the surface.
 25. The method of claim 24,wherein: each of the probe locations has an area of one-hundredth of asquare centimeter or less.
 26. The method of claim 1, wherein: thesecond substrate comprises more than one thousand different ligands ofknown sequence collectively occupying an area of less than one squarecentimeter, the different ligands occupying different known locationswithin the area.
 27. The method of claim 1, wherein: the secondsubstrate has a surface comprising more than ten different nucleic acidsof known sequences at a plurality of probe locations on the surface ofthe second substrate, each of the probe locations having an area ofone-hundredth of a square centimeter or less, at least one of thenucleic acids being bound to a labeled receptor.
 28. The method of claim1, wherein: the at least one filtered excitation beam is a laser beam.29. The method of claim 1, wherein: the plurality of pixel locationscomprises one or more scan lines.
 30. The method of claim 1, wherein:the representative excitation values are each related to a power of theexcitation beam.
 31. The method of claim 1, wherein: the emission signalarises from the direction of the excitation beam to the plurality ofpixel locations.